Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Aug;63(8):3015–3020. doi: 10.1128/iai.63.8.3015-3020.1995

Analysis of a second bacteriophage hyaluronidase gene from Streptococcus pyogenes: evidence for a third hyaluronidase involved in extracellular enzymatic activity.

W L Hynes 1, L Hancock 1, J J Ferretti 1
PMCID: PMC173410  PMID: 7622224

Abstract

The hyaluronidase gene (hylP2) from a second group A streptococcal bacteriophage was isolated from ATCC T-type-22 hyaluronidase-producing strain 10403, a strain known to produce increased amounts of extracellular hyaluronidase. Sequence analysis of hylP2 and alignment with the previously described bacteriophage hyaluronidase gene (hylP) showed a high degree of similarity; however, hylP2 had deletions of regions specifying 34 amino acids. Twenty-eight of the deleted amino acids were in a region of HylP containing a series of collagen-like Gly-X-Y repeating units. By employing primers for both hylP and hylP2, PCR amplification resulted in fragments of appropriate sizes in 97% of the strains tested, with some strains producing two fragments, indicating the presence of at least two phages. When the hylP2 gene was introduced via a plasmid vector into a non-hyaluronidase-producing Streptococcus pyogenes strain, this strain was still unable to produce extracellular hyaluronidase, although intracellular hyaluronidase was present. These results, along with the absence of a typical N-terminal signal peptide, indicate that HylP2 is unable to be secreted into the extracellular milieu. Examination of more than 100 strains for production of hyaluronidase showed that only 23% of the strains produced extracellular hyaluronidase. One of these strains (strain 10403) contains a single bacteriophage hyaluronidase gene (hylP2) which, when inactivated by allelic replacement, still produces large amounts of extracellular hyaluronidase. These results suggest the presence of a different hyaluronidase gene encoding a protein that is actively secreted into the extracellular milieu.

Full Text

The Full Text of this article is available as a PDF (322.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Benchetrit L. C., Avelino C. C., Barrucand L., Sá Figueiredo A. M., de Oliveira C. M. Hyaluronidase production by groups A, B, C, and G streptococci: a statistical analysis. Zentralbl Bakteriol Mikrobiol Hyg A. 1984 May;257(1):27–37. [PubMed] [Google Scholar]
  2. Benchetrit L. C., Gray E. D., Edstrom R. D., Wannamaker L. W. Purification and characterization of a hyaluronidase associated with a temperate bacteriophage of group A, type 49 streptococci. J Bacteriol. 1978 Apr;134(1):221–228. doi: 10.1128/jb.134.1.221-228.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Benchetrit L. C., Gray E. D., Wannamaker L. W. Hyaluronidase activity of bacteriophages of group A streptococci. Infect Immun. 1977 Feb;15(2):527–532. doi: 10.1128/iai.15.2.527-532.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benchetrit L. C., Wannamaker L. W., Gray E. D. Immunological properties of hyaluronidases associated with temperate bacteriophages of group A streptococci. J Exp Med. 1979 Jan 1;149(1):73–83. doi: 10.1084/jem.149.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berry A. M., Lock R. A., Thomas S. M., Rajan D. P., Hansman D., Paton J. C. Cloning and nucleotide sequence of the Streptococcus pneumoniae hyaluronidase gene and purification of the enzyme from recombinant Escherichia coli. Infect Immun. 1994 Mar;62(3):1101–1108. doi: 10.1128/iai.62.3.1101-1108.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Canard B., Garnier T., Saint-Joanis B., Cole S. T. Molecular genetic analysis of the nagH gene encoding a hyaluronidase of Clostridium perfringens. Mol Gen Genet. 1994 Apr;243(2):215–224. doi: 10.1007/BF00280319. [DOI] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fiszer-Szafarz B. Hyaluronidase polymorphism detected by polyacrylamide gel electrophoresis. Application to hyaluronidases from bacteria, slime molds, bee and snake venoms, bovine testes, rat liver lysosomes, and human serum. Anal Biochem. 1984 Nov 15;143(1):76–81. doi: 10.1016/0003-2697(84)90560-8. [DOI] [PubMed] [Google Scholar]
  9. GREILING H., STUHLSATZ H. W., EBERHARD T. ZUR HETEROGENITAET DER HYALURONATLYASE. Hoppe Seylers Z Physiol Chem. 1965;340:243–248. doi: 10.1515/bchm2.1965.340.1-2.243. [DOI] [PubMed] [Google Scholar]
  10. Greiling H., Stuhlsatz H. W., Eberhard T., Eberhard A. Studies on the mechanism of hyaluronate lyase action. Connect Tissue Res. 1975;3(2):135–139. doi: 10.3109/03008207509152171. [DOI] [PubMed] [Google Scholar]
  11. Halperin S. A., Ferrieri P., Gray E. D., Kaplan E. L., Wannamaker L. W. Antibody response to bacteriophage hyaluronidase in acute glomerulonephritis after group A streptococcal infection. J Infect Dis. 1987 Feb;155(2):253–261. doi: 10.1093/infdis/155.2.253. [DOI] [PubMed] [Google Scholar]
  12. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  13. Herd J. K., Motycka L. Detection of proteolytic enzymes in agar electrophoresis. Anal Biochem. 1973 Jun;53(2):514–521. doi: 10.1016/0003-2697(73)90101-2. [DOI] [PubMed] [Google Scholar]
  14. Huang T. T., Malke H., Ferretti J. J. Heterogeneity of the streptokinase gene in group A streptococci. Infect Immun. 1989 Feb;57(2):502–506. doi: 10.1128/iai.57.2.502-506.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hynes W. L., Ferretti J. J. Assays for hyaluronidase activity. Methods Enzymol. 1994;235:606–616. doi: 10.1016/0076-6879(94)35174-0. [DOI] [PubMed] [Google Scholar]
  16. Hynes W. L., Ferretti J. J., Gilmore M. S., Segarra R. A. PCR amplification of streptococcal DNA using crude cell lysates. FEMS Microbiol Lett. 1992 Jul 1;73(1-2):139–142. doi: 10.1016/0378-1097(92)90597-h. [DOI] [PubMed] [Google Scholar]
  17. Hynes W. L., Ferretti J. J. Sequence analysis and expression in Escherichia coli of the hyaluronidase gene of Streptococcus pyogenes bacteriophage H4489A. Infect Immun. 1989 Feb;57(2):533–539. doi: 10.1128/iai.57.2.533-539.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. KJEMS E. Studies on streptococcal bacteriophages. 2. Adsorption, lysogenization, and one-step growth experiments. Acta Pathol Microbiol Scand. 1958;42(1):56–66. [PubMed] [Google Scholar]
  19. KJEMS E. Studies on streptococcal bacteriophages. 5. Serological investigation of phages isolated from 91 strains of group A haemolytic streptococci. Acta Pathol Microbiol Scand. 1960;49:205–212. [PubMed] [Google Scholar]
  20. Lin B., Hollingshead S. K., Coligan J. E., Egan M. L., Baker J. R., Pritchard D. G. Cloning and expression of the gene for group B streptococcal hyaluronate lyase. J Biol Chem. 1994 Dec 2;269(48):30113–30116. [PubMed] [Google Scholar]
  21. Niemann H., Birch-Andersen A., Kjems E., Mansa B., Stirm S. Streptococcal bacteriophage 12/12-borne hyaluronidase and its characterization as a lyase (EC 4.2.99.1) by means of streptococcal hyaluronic acid and purified bacteriophage suspensions. Acta Pathol Microbiol Scand B. 1976 Jun;84(3):145–153. doi: 10.1111/j.1699-0463.1976.tb01917.x. [DOI] [PubMed] [Google Scholar]
  22. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Simon D., Chopin A. Construction of a vector plasmid family and its use for molecular cloning in Streptococcus lactis. Biochimie. 1988 Apr;70(4):559–566. doi: 10.1016/0300-9084(88)90093-4. [DOI] [PubMed] [Google Scholar]
  24. Simon D., Ferretti J. J. Electrotransformation of Streptococcus pyogenes with plasmid and linear DNA. FEMS Microbiol Lett. 1991 Aug 1;66(2):219–224. doi: 10.1016/0378-1097(91)90336-9. [DOI] [PubMed] [Google Scholar]
  25. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  26. Stern M., Stern R. A collagenous sequence in a prokaryotic hyaluronidase. Mol Biol Evol. 1992 Nov;9(6):1179–1180. doi: 10.1093/oxfordjournals.molbev.a040786. [DOI] [PubMed] [Google Scholar]
  27. Trieu-Cuot P., Carlier C., Poyart-Salmeron C., Courvalin P. A pair of mobilizable shuttle vectors conferring resistance to spectinomycin for molecular cloning in Escherichia coli and in gram-positive bacteria. Nucleic Acids Res. 1990 Jul 25;18(14):4296–4296. doi: 10.1093/nar/18.14.4296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wannamaker L. W., Skjold S., Maxted W. R. Characterization of bacteriophages from nephritogenic group A streptococci. J Infect Dis. 1970 Apr;121(4):407–418. doi: 10.1093/infdis/121.4.407. [DOI] [PubMed] [Google Scholar]
  29. Wessels M. R., Goldberg J. B., Moses A. E., DiCesare T. J. Effects on virulence of mutations in a locus essential for hyaluronic acid capsule expression in group A streptococci. Infect Immun. 1994 Feb;62(2):433–441. doi: 10.1128/iai.62.2.433-441.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wessels M. R., Moses A. E., Goldberg J. B., DiCesare T. J. Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8317–8321. doi: 10.1073/pnas.88.19.8317. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES